Venous malformation (VM) is a chronic condition often seen at birth or during childhood. VMs are lesions composed by widened, abnormally shaped veins. Smooth muscle cells are absent in many areas. The lesion tends to enlarge with age and can suddenly expand causing deformity and/or pain as a result of clot formation. No targeted therapies are available, and treatments for VM are very limited, including only sclerotherapy and reconstructive surgery. After treatment, lesions often recur. Germline or somatic TIE2 mutations have been associated with VMs. TIE2 is an endothelial receptor tyrosine kinase that regulates both maintenance of vascular quiescence and promotion of angiogenesis. Our hypothesis is that TIE2 mutations in the endothelial cells cause aberrant venous development due to an incorrect differentiation and/or recruitment of perivascular cells. Therefore we propose to use endothelial cells expressing the TIE2 mutations found in patients to recapitulate the VM phenotype in mouse. A murine model of VM has never been reported. Our preliminary data demonstrate that HUVECs expressing TIE2-L914F mutated receptor, when injected subcutaneously in mouse, form vascular lesions that tend to grow over time. The histology reveals excessively widened blood-filled vessels. Perivascular cells around the abnormal channels are scarce in number, and blood flow in the lesion is slow and mainly non-pulsatile. As control we used HUVECs expressing TIE2-Wild Type (WT), these cells are incapable of forming blood vessels in our model. Injection of normal ECs in combination with bone marrow mesenchymal progenitor cells (bmMPC) results in formation of regular sized blood vessels. HUVECs TIE2-L914F injected in combination with bmMPC formed instead vessels with enlarged lumen, with scarce perivascular cells, and no evident differentiation/maturation of bmMPC. HUVECs TIE2-WT behaved similarly to normal ECs. We thereby hypothesize that the TIE2 mutation inhibits perivascular cells differentiation, and the correct control of the lumen siz. Our research will focus on: Aim 1- Establishing a VM-murine model that reflects the patient's disease, with the use of EC with mutated TIE2 receptor or cells isolated from patient VMs; Aim 2- Determine the effects of TIE2 mutations in the perivascular cell differentiation, control of lumen size, and motility we will use bmMPC, pericytes or SMCs, in the murine VM model and in 3D collagen in vitro model; Aim 3- Perform pre-clinical studies to identify a novel targeted treatment for VMs, in order to normalize or induce regression of the pathological vasculature and avoid a rebound of the disease.